Molecular Biology of the CellVol. 21, 2624–2638, August 1, 2010

Defects in the Secretory Pathway and High Ca2� InduceMultiple P-bodiesCornelia Kilchert, Julie Weidner, Cristina Prescianotto-Baschong, and Anne Spang

Biozentrum, Growth and Development, University of Basel, CH-4056 Basel, Switzerland

Submitted February 4, 2010; Accepted May 25, 2010Monitoring Editor: Reid Gilmore

mRNA is sequestered and turned over in cytoplasmic processing bodies (PBs), which are induced by various cellularstresses. Unexpectedly, in Saccharomyces cerevisiae, mutants of the small GTPase Arf1 and various secretory pathwaymutants induced a significant increase in PB number, compared with PB induction by starvation or oxidative stress.Exposure of wild-type cells to osmotic stress or high extracellular Ca2� mimicked this increase in PB number. Conversely,intracellular Ca2�-depletion strongly reduced PB formation in the secretory mutants. In contrast to PB induction throughstarvation or osmotic stress, PB formation in secretory mutants and by Ca2� required the PB components Pat1 and Scd6,and calmodulin, indicating that different stressors act through distinct pathways. Consistent with this hypothesis, whenstresses were combined, PB number did not correlate with the strength of the translational block, but rather with the typeof stress encountered. Interestingly, independent of the stressor, PBs appear as spheres of �40–100 nm connected to theendoplasmic reticulum (ER), consistent with the idea that translation and silencing/degradation occur in a spatiallycoordinated manner at the ER. We propose that PB assembly in response to stress occurs at the ER and depends onintracellular signals that regulate PB number.

INTRODUCTION

Cells adapt to stress by varying their proteome. Thesechanges in protein expression can be achieved through tran-scriptional and translational control or through changes inprotein stability. Stress causes attenuation of general trans-lation, whereas the translation of a subset of mRNAs isup-regulated. Many mRNAs are sequestered in processingbodies or P-bodies (PBs) or stress granules (SGs) in responseto stress. In SGs, mRNAs are stored until the stress is alle-viated and the mRNAs can return to the cytosol (Coller andParker, 2005). PBs on the other hand, are sites of mRNAstorage and turnover. The stress conditions that result ineither PB or SG formation are only partially overlapping.Recent evidence suggests that PB formation could precedestress granule formation, and PBs could mature either intostress granules or into mRNA-degrading PBs (Buchan et al.,2008). However, SGs could potentially also form indepen-dently from PBs. Although the mechanism of SG assemblystill remains elusive, more is known about PB assembly.According to the current model, two separate complexesbind the mRNA: the decapping complex at the 5� end andthe Lsm–Pat1 complex at the 3� end of the mRNAs, topromote interaction between different mRNPs to allow PBassembly (Decker et al., 2007; Franks and Lykke-Andersen,2008; Reijns et al., 2008). Thus, loss of one complex mayreduce the efficiency with which PBs are formed. In yeast,the decapping complex contains the decapping proteinsDcp1 and Dcp2 and the decapping promoting factor Edc3.The 3�-binding Lsm–Pat1 complex consists of Sm and Sm-like (Lsm) proteins, which form two heptameric rings that

encircle the RNA (Salgado-Garrido et al., 1999) and to whichthe decapping activator Pat1 is recruited. The Lsm–Pat1complex shows an inherent affinity to deadenylated mRNAsequences (Bouveret et al., 2000; Tharun et al., 2000, 2005;Tharun and Parker, 2001; Chowdhury et al., 2007). PB for-mation seems to correlate with defects in translation initia-tion, whereas translation elongation problems do not causePBs to form (Eulalio et al., 2007; Parker and Sheth, 2007).Despite what is known about PB assembly, it is still debatedif PBs are merely aggregations of “unused” mRNA orwhether PB assembly is regulated through distinct signals.

Components of the secretory pathway are responsible forthe transport of proteins and lipids between cellular com-partments as well as to the cell surface. Intracellular traffick-ing components have been implicated in mRNA transport(Aronov and Gerst, 2004; Trautwein et al., 2004; Bi et al.,2007). Interestingly, Deloche et al. (2004) showed that inyeast at least a subset of secretory transport mutants failed toproperly initiate translation. This translation attenuation islikely a consequence of membrane stress caused by blocksalong the secretory pathway. Given that blocked translationinitiation can lead to PB formation (Eulalio et al., 2007;Parker and Sheth, 2007), we asked whether mutants in thesecretory pathway also promote PB formation in S. cerevisiae.We found that many more PBs were formed in secretorytransport mutants than after induction of PBs upon starva-tion in wild-type cells. The PBs observed in secretory mu-tants and upon starvation were indistinguishable by size orby Dcp2-myc and Dhh1-myc content, as judged by immu-noelectron microscopy. Dcp2-myc and Dhh1-myc formedsphere-like structures 40–100 nm in diameter. The multiplePB phenotype was also induced in wild-type cells by theapplication of hyperosmotic shock or by increasing extracel-lular Ca2� levels. The induction of numerous PBs in re-sponse to membrane stress required functional calmodulinand the PB components Scd6 and Pat1. Interestingly, muta-tions in calmodulin or deletion of PAT1, or SCD6, did not

This article was published online ahead of print in MBoC in Press(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E10–02–0099)on June 2, 2010.

Address correspondence to: Anne Spang (anne.spang@unibas.ch).

2624 © 2010 by The American Society for Cell Biology http://www.molbiolcell.org/content/suppl/2010/06/02/E10-02-0099.DC1Supplemental Material can be found at:

interfere with PB induction through starvation or hyperos-motic shock. The effect of inducing PBs under starvation andCa2� was additive because the block in translation initiationwas much stronger when stresses were combined, but nev-ertheless multiple PBs were induced. Our results demon-strate that distinct signaling pathways are in place to inducePB production, depending on the stress encountered, andhence PB formation is more than just the mere consequenceof a block in translation initiation. In this study we uncoverthat PB assembly is not the result of aggregation, but isinduced through distinct pathways, one of which requirescalmodulin.

MATERIALS AND METHODS

Yeast MethodsStandard genetic techniques were used throughout (Sherman, 1991). Allmodifications were carried out chromosomally, with the exceptions listedbelow. Chromosomal tagging and deletions were performed as described inKnop et al. (1999) and Gueldener et al. (2002). The temperature-sensitivealleles sec21-1, sec27-1, and cmd1-3 were transferred into the YPH499 orNYY0-1 background according to Erdeniz et al. (1997). The cmd1-3 allele wasintroduced into the arf1-11 background using plasmid pHS47 (URA3) thatwas kindly provided by E. Schiebel (University of Heidelberg, Heidelberg,Germany). After plasmid transformation the wild-type allele CMD1 wasdeleted on the chromosome. The sec6-4 allele was integrated into the SEC6locus using the pRS406 vector (Sikorski and Hieter, 1989). The sar1-D32Gallele was introduced into YPH499 on plasmid pMYY3–1 (TRP1) that was agift from A. Nakano (RIKEN, Saitama, Japan). For ER costaining, cells weretransformed with pSM1959 (LEU2) or pSM1960 (URA3), high-copy plasmidscarrying Sec63-RFP, which were kindly provided by S. Michaelis (JohnsHopkins University, Baltimore, MD). Strains used are listed in Table 1.

Fluorescence MicroscopyYeast cells were grown in YPD to early log phase and shifted for 1 h to 37°Cor subjected to various stresses where indicated; arf1-11 �slt2 required addi-tional osmotic support for growth and was cultured in medium containing 1M sorbitol. The cells were taken up in HC-complete medium (without glucoseor supplemented with CaCl2 or NaCl, where indicated) and immobilized onconcanavalin A–coated slides. Fluorescence was monitored with an Axiocammounted on an Axioplan 2 fluorescence microscope (Carl Zeiss, Oberkochen,Germany) using Axiovision software. Image processing was performed usingAdobe Photoshop CS2 (San Jose, CA). For counting, pictures were exported toPhotoshop and inverted, and the tonal range was adjusted using the levelsdialog box to facilitate counting; all pictures from the same experiment weretreated equally. A minimum of 100 cells from at least two independentexperiments was counted for each condition. In the quantification graphs, thesize of the box is determined by the 25th and 75th percentiles, the whiskersrepresent the 5th and 95th percentiles, the horizontal line and the little squaremark the median and the mean, respectively.

Denaturing Yeast Extracts and Western BlotFifteen milliliters of yeast culture was grown to early log phase (OD6000.5–0.7) and shifted for 1 h to 37°C where indicated. The cells were harvestedand lysed with glass beads in 150 �l of lysis buffer (20 mM Tris/HCl, pH 8.0,5 mM EDTA) in the presence of 1 mM dithiothreitol (DTT) and proteaseinhibitors. The lysates were incubated at 65°C for 5 min, and unlysed cellssubsequently were removed by centrifugation. The protein concentration wasdetermined using the DC Protein Assay (Bio-Rad, Richmond, CA), and theequivalent of 30 �g of total protein was analyzed by SDS-PAGE and immu-noblotting. Total Slt2 was detected using goat anti-Mpk1 antibody (yN-19,Santa Cruz Biotechnology, Santa Cruz, CA) and phospho-Slt2 using rabbitantiphospho-p44/42 MAP kinase (Thr202/Tyr204) antibody (Cell Signaling,Beverly, MA) with horseradish peroxidase-conjugated secondary antibodies(Pierce, Rockford, IL) and enhanced chemiluminescence reagent (GE Health-care, Freiburg, Germany).

Polysome Profile AnalysisPolysome preparations were performed as described previously (de la Cruz etal., 1997) on 4–47% sucrose gradients prepared with a Gradient Master(Nycomed Pharma, Westbury, NY). Gradient analysis was performed using agradient fractionator (Labconco, Kansas City, MO) and the Acta FPLC system(GE Healthcare) and continuously monitored at A254.

Immunoelectron MicroscopyCells expressing Dcp2–9myc or Dhh1–9myc were grown to early log-phase at23°C and then shifted to 37°C for 1 h or transferred to a medium lacking a

carbon source for 15 min. Cells were fixed and treated for immunoelectronmicroscopy as described in Prescianotto-Baschong and Riezman (2002). Ten-nanometer gold particles coupled to goat anti-rabbit IgG (BBInternational,Cardiff, United Kingdom) were used to detect the binding of polyclonal rabbitanti-myc antibodies (Abcam, Cambridge, MA).

Flotation of PBsFlotation of ER membranes was performed according to Schmid et al. (2006).The equivalent of 50 OD600 units was converted into spheroplasts at 37°C andlysed by Dounce homogenization in 3 ml of lysis buffer (20 mM HEPES/KOH, pH 7.6, 100 mM sorbitol, 100 mM KAc, 5 mM Mg(Ac)2, 1 mM EDTA,100 �g/ml cycloheximide) in the presence of 1 mM DTT and protease inhib-itors. After removal of cellular debris (5 min, 300 � g), membranes werepelleted by centrifugation (10 min, 13,000 � g), resuspended in 2 ml of lysisbuffer containing 50% sucrose, and layered on top of 2 ml 65% sucrose in lysisbuffer. Two additional 5- and 2-ml cushions (40 and 0% sucrose) were layeredon top. The step gradient was spun in a TST41.14 rotor for 16 h at 28,000 � g.After centrifugation, 1-ml fractions were collected from each of the cushionsand the interphases and were TCA-precipitated. The samples were analyzedby SDS-PAGE and immunoblotting using monoclonal mouse anti-myc anti-bodies (9E10, Sigma, St. Louis, MO) and polyclonal rabbit anti-Sec61 antibod-ies (a gift from R. Schekman, University of California at Berkeley, Berkeley,CA).

Northern BlotYeast cells were grown in YPD to early log phase and shifted for 1 h to 37°Cwhere indicated. Cells were lysed in lysis buffer by grinding in liquid nitro-gen. RNA was extracted from the P13 pellet using TriZOL reagent (Invitro-gen, Carlsbad, CA), and 15 �g of RNA was resolved on agarose gels contain-ing formaldehyde. The RNA was transferred onto Hybond N� (AmershamBiosciences, Piscataway, NJ) and subsequently hybridized to HAC1 probes,which were generated using an AlkPhos direct labeling kit (Amersham Bio-sciences). The probes were detected using the CDP-Star reagent (GE Health-care) according to manufacturer’s recommendations.

RESULTS

PB Number Is Increased in arf1 MutantsSeveral mutants in the secretory pathway lead to attenuationof translation (Deloche et al., 2004). In addition, specificmutant alleles of the small GTPase Arf1 are defective in theasymmetric distribution of ASH1 mRNA, and these defectsare not caused by disturbances of the actin cytoskeleton(Trautwein et al., 2004). Therefore, we wondered whetherarf1 mutants induce PBs, which provide a storage and deg-radation location for mRNAs in response to translationalarrest. As a marker for PBs we used Dcp2 (decapping pro-tein 2), which is required for the decapping of mRNAs andfor PB formation (Dunckley and Parker, 1999; Sheth andParker, 2003; Teixeira and Parker, 2007). We chromosomallyappended Dcp2 with green fluorescent protein (GFP) anddetermined the number of PBs in control and temperature-sensitive arf1 mutant cells (Figure 1A). As expected, few PBswere observed in wild-type cells or in arf1 mutants at thepermissive temperature, with Dcp2-GFP largely distributedthroughout the cytosol. Strikingly, a large increase in PBnumber (9–10 on average) was observed in arf1 mutantalleles upon shift to 37°C (Figure 1, A and B). The temper-ature shift represents considerable stress for the wild type,but does not induce a block in translation, and only 1–2 PBswere present in wild-type cells at 37°C (Figure 1, A and B).

We have previously shown that arf1-11 and arf1-18 but notarf1-17 failed to localize ASH1 mRNA to the bud tip of yeastcells (Trautwein et al., 2004). Strikingly, the arf1-17 mutationalso caused a dramatic increase in PB number similar to thatdetected in arf1-11 and arf1-18, indicating that mislocaliza-tion of mRNAs that are dependent on the SHE machinery isnot the cause of multiple PB formation (Figure 1, A and B).

The Dcp2 foci we observed in arf1 mutants likely represent Pbodies and not stress granules (or EGP-bodies) because, gen-erally, stress granules do not contain Dcp2 (Kedersha et al.,2005; Anderson et al., 2006; Hoyle et al., 2007). To provide

Ca2�-induced P-body Formation

Vol. 21, August 1, 2010 2625

Table 1. Yeast strains

Strain Designation Genotype Reference

YPH499 WT MAT a ade2-101 his3-200 leu2-1 lys2-801 trp-63 ura3-52 Sikorski and Hieter(1989)

NYY0-1 ARF1 MAT a ade2::ARF1::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3 leu2 Yahara et al. (2001)NYY11-1 arf1-11 MAT a ade2::arf1-11::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3

leu2Yahara et al. (2001)

NYY17-1 arf1-17 MAT a ade2::arf1-17::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2

Yahara et al. (2001)

NYY18-1 arf1-18 MAT a ade2::arf1-18::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2

Yahara et al. (2001)

YAS1031A ARF1 Dcp2-GFP MAT a ade2::ARF1::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3 leu2DCP2::DCP2-yEGFP-kanMX4

This study

YAS1032A arf1-11 Dcp2-GFP MAT a ade2::arf1-11::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4

This study

YAS1033A arf1-17 Dcp2-GFP MAT a ade2::arf1-17::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4

This study

YAS1034A arf1-18 Dcp2-GFP MAT a ade2::arf1-18::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4

This study

YAS2428 ARF1 Edc3-eqFP611eIFG2-GFP

MAT a ade2::ARF1::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3 leu2EDC3::EDC3-eqFP611-kanMX4 eIFG2::eIFG2-yEGFP-TRP1 (K. lactis)

This study

YAS2429 arf1-11 Edc3-eqFP611eIFG2-GFP

MAT a ade2::arf1-11::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2 EDC3::EDC3-eqFP611-kanMX4 eIFG2::eIFG2-yEGFP-TRP1 (K.lactis)

This study

YAS1681 sar1-D32G Dcp2-GFP MAT a ade2-101 his3-200 leu2-1 lys2-801 trp-63 ura3-52 SAR1::LEU2(K. lactis) pMYY3-1(Ycp�sar1-D32G TRP1�)DCP2::DCP2-yEGFP-kanMX4

This study

YAS1133 sec21-1 Dcp2-GFP MAT � ade2-101 his3-200 leu2-1 lys2-801 trp-63 ura3-52 sec21-1DCP2::DCP2-yEGFP-kanMX4

This study

YAS1134 sec27-1 Dcp2-GFP MAT a ade2-101 his3-200 leu2-1 lys2-801 trp-63 ura3-52 sec27-1DCP2::DCP2-yEGFP-kanMX4

This study

YAS1131 �gea2 gea1-19 Dcp2-GFP

MAT a ura3-52 leu2,3-112 his3-200 GEA2::HIS3 gea1-19DCP2::DCP2-yEGFP-kanMX4

This study

YAS1135 sec6-4 Dcp2-GFP MAT a ade2-101 his3-200 leu2-1 lys2-801 trp-63 ura3-52 sec6-4CHS6::CHS6-9myc-TRP1 (K. lactis) DCP2::DCP2-yEGFP-kanMX4

This study

YAS1877 sec3-2 Dcp2-GFP MAT a ura3 leu2 trp1 sec3-2 DCP2::DCP2-yEGFP-kanMX4 This studyYAS1880 sec4-8 Dcp2-GFP MAT a ura3 leu2 lys sec4-8 DCP2::DCP2-yEGFP-kanMX4 This studyYAS1878 sec2-41 Dcp2-GFP MAT a ura3 leu2 his3 lys2 trp1 ade2 sec2-41

DCP2::DCP2-yEGFP-kanMX4This study

YAS2235 ARF1 Pub1-GFP MAT a ade2::ARF1::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3 leu2PUB1::PUB1-yEGFP-kanMX4

This study

YAS1945 ARF1 Dcp2-GFP�hog1

MAT a ade2::ARF1::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 HOG::LEU2 (K . lactis)

This study

YAS1946 arf1-11 Dcp2-GFP�hog1

MAT a ade2::arf1-11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 HOG::LEU2 (K. lactis)

This study

YAS1685 ARF1 Dcp2-GFP�sho1

MAT a ade2::ARF1::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 SHO1::LEU2 (K. lactis)

This study

YAS1686 arf1-11 Dcp2-GFP�sho1

MAT a ade2::arf1-11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 SHO1::LEU2 (K. lactis)

This study

YAS1947 ARF1 Dcp2-GFP�sko1

MAT a ade2::ARF1::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 SKO1::LEU2 (K. lactis)

This study

YAS1948 arf1-11 Dcp2-GFP�sko1

MAT a ade2::arf1-11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 SKO1::LEU2 (K. lactis)

This study

YAS1967 ARF1 Dcp2-GFP �slt2 MAT a ade2::ARF1::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 SLT2::LEU2 (K. lactis)

This study

YAS1968 arf1-11 Dcp2-GFP�slt2

MAT a ade2::arf1-11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 SLT2::LEU2 (K. lactis)

This study

YAS1986 ARF1 Dcp2-GFP�hog1 �slt2

MAT a ade2::ARF11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 HOG1::LEU2 (K. lactis)SLT2::URA3 (K. lactis)

This study

YAS1987 arf1-11 Dcp2-GFP�hog1 �slt2

MAT a ade2::arf1-11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 HOG1::LEU2 (K. lactis)SLT2::URA3 (K. lactis)

This study

YAS2092 ARF1 Dcp2-GFP�hog1 �slt2 �cnb1

MAT a ade2::ARF1::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 HOG1::loxP SLT2::loxPCNB1::LEU2 (K. lactis)

This study

YAS2292 arf1-11 Dcp2-GFP�hog1 �slt2 �cnb1

MAT a ade2::arf1-11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 HOG1::loxP SLT2::loxPCNB1::LEU2 (K. lactis)

This study

(Continued)

C. Kilchert et al.

Molecular Biology of the Cell2626

corroborating evidence we appended another PB component,the helicase Dhh1, with GFP, which behaved similarly to Dcp2-GFP in the arf1 strains (Supplemental Figure 1). Moreover,

deletion of an essential SG component, PUB1, (Buchan et al.,2008; Swisher and Parker, 2010) did not interfere with theformation of Dcp2-GFP-positive structures (Supplemental Fig-

Table 1. Continued

Strain Designation Genotype Reference

YAS2575 ARF1 Dcp2-GFP�ypk1

MAT a ade2::ARF1::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 YPK1::LEU2 (K. lactis)

This study

YAS2010 arf1-11 Dcp2-GFP�ypk1

MAT a ade2::arf1-11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 YPK1::LEU2 (K. lactis)

This study

YAS2021 ARF1 Dcp2-GFP�cnb1

MAT a ade2::ARF11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 CNB1::LEU2 (K. lactis)

This study

YAS2051 arf1-11 Dcp2-GFP�cnb1

MAT a ade2::arf1-11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 CNB1::LEU2 (K. lactis)

This study

YAS2088 ARF1 Dcp2-GFP�cmk1 �cmk2

MAT a ade2::ARF11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 CMK1::LEU2 (K. lactis)CMK2::URA3 (K. lactis)

This study

YAS2089 arf1-11 Dcp2-GFP�cmk1 �cmk2

MAT a ade2::arf1-11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 CMK1::LEU2 (K. lactis)CMK2::URA3 (K. lactis)

This study

YAS2090 ARF1 Dcp2-GFP�cmk1 �cmk2 �cnb1

MAT a ade2::ARF1::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 CMK1::loxP CMK2::loxPCNB1::LEU2 (K. lactis)

This study

YAS2091 arf1-11 Dcp2-GFP�cmk1 �cmk2 �cnb1

MAT a ade2::arf1-11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 CMK1::loxP CMK2::loxPCNB1::LEU2 (K. lactis)

This study

YAS2586 arf1-11 Dcp2-GFP�gcn2

MAT a ade2::arf1-11::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 GCN2::URA3 (K. lactic)

This study

YAS2588 arf1-11 Dcp2-GFP�ire1

MAT a ade2::arf1-11::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 IRE1::URA3 (K. lactic)

This study

YAS2296 ARF1 cmd1-3 Dcp2-GFP

MAT a ade2::ARF1::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 CMD1::cmd1-3

This study

YAS2580 arf1-11 cmd1-3 Dcp2-GFP

MAT a ade2::arf1-11::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4

This study

pHS47(cmd1-3 URA3) CMD1::kanMX4YAS2294 ARF1 Dcp2-GFP

�pat1MAT a ade2::ARF1::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3

leu2 DCP2::DCP2-yEGFP-kanMX4 PAT1::LEU2 (K. lactis)This study

YAS2295 arf1-11 Dcp2-GFP�pat1

MAT a ade2::arf1-11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 PAT1::LEU2 (K. lactis)

This study

YAS2297 sec6-4 Dcp2-GFP�pat1

MAT a ade2-101 his3-200 leu2-1 lys2-801 trp-63 ura3-52 sec6-4CHS6::CHS6-9myc-TRP1 (K. lactis) DCP2::DCP2-yEGFP-kanMX4PAT1::LEU2 (K. lactis)

This study

YAS1097 ARF1 Dcp2-GFP�scd6

MAT a ade2::ARF1::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3 leu2DCP2::DCP2-yEGFP-kanMX4 SCD6::LEU2 (K. lactis)

This study

YAS1098 arf1-11 Dcp2-GFP�scd6

MAT a ade2::arf1-11::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-yEGFP-kanMX4 SCD6::LEU2 (K. lactis)

This study

YAS2500 sec6-4 Dcp2-GFP�scd6

MAT a ade2-101 his3-200 leu2-1 lys2-801 trp-63 ura3-52 sec6-4CHS6::CHS6-9myc-TRP1 (K. lactis) DCP2::DCP2-yEGFP-kanMX4SCD6::LEU2 (K. lactis)

This study

YAS1294 ARF1 Dcp2-9myc MAT a ade2::ARF1::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3 leu2DCP2::DCP2-9myc-TRP1 (K. lactis)

This study

YAS1295 arf1-11 Dcp2-9myc MAT a ade2::arf1-11::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2 DCP2::DCP2-9myc-TRP1 (K. lactis)

This study

YAS1693 ARF1 Dhh1-9myc MAT a ade2::ARF1::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DHH1::DHH1-9myc-TRP1 (K. lactis)

This study

YAS1694 arf1-11 Dhh1-9myc MAT a ade2::arf1-11::ADE2 ARF1::HIS3 ARF2::HIS3 ura3 lys2 trp1 his3leu2 DHH1::DHH1-9myc-TRP1 (K. lactis)

This study

YAS2576 arf1-11 Pat1-GFP MAT a ade2::arf1-11::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2 PAT1::PAT1-yEGFP-TRP1 (K. lactis)

This study

YAS2578 arf1-11 Scd6-GFP MAT a ade2::arf1-11::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2 SCD6::SCD6-yEGFP-kanMX4

This study

YAS1153 ARF1 Dhh1-GFP MAT a ade2::ARF1::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3 leu2DHH1::DHH1-yEGFP-kanMX4

This study

YAS1154 arf1-11 Dhh1-GFP MAT a ade2::arf1-11::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2 DHH1::DHH1-yEGFP-kanMX4

This study

YAS2153 ARF1 Cmd1-GFP MAT a ade2::ARF1::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3 leu2CMD1::CMD1-yEGFP-kanMX4

This study

YAS2154 arf1-11 Cmd1-GFP MAT a ade2::arf1-11::ADE2 arf1::HIS3 arf2::HIS3 ura3 lys2 trp1 his3leu2 CMD1::CMD1-yEGFP-kanMX4

This study

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ure 2A). Finally, the SG marker eIF4G2 fused to GFP did notaccumulate in foci in arf1-11 at 37°C (Figure 1C).

PB Number Is Increased in a Variety of SecretoryTransport MutantsBecause the major function of Arf1p is to initiate coat proteinI (COPI)- and clathrin-coated vesicle budding events, we

asked whether the increase in PBs is a common feature insecretory transport mutants. We monitored Dcp2-GFP invarious temperature-sensitive secretory transport mutantscovering endoplasmic reticulum (ER)3Golgi, Golgi3ER,and post-Golgi trafficking steps and determined the numberof PBs after a shift for 1 h to the nonpermissive temperature(Figure 1D). Interestingly, most of the mutants showed an

Figure 1. arf1 and secretory pathway mutants have multiple PBs. (A) The PB marker Dcp2 was chromosomally tagged with GFP in thecontrol strain and in several temperature-sensitive arf1 mutants. At the permissive temperature (23°C), no PBs are observed andDcp2-GFP is dispersed throughout the cytoplasm; upon shift to the nonpermissive temperature (37°C) for 1 h, PB formation is inducedin all strains. The increase in PB number is more pronounced in arf1 mutants than in the control. (B) Quantification of the multiple PBphenotype in arf1 mutants at nonpermissive temperature. A minimum of a hundred cells from at least two independent experimentswas counted for each condition. The size of the box is determined by the 25th and 75th percentiles; the whiskers represent the 5th and95th percentiles, the horizontal line and the little square mark the median and the mean, respectively. (C) Wild-type and arf1-11 mutantcells expressing the PB marker Edc3-eqFP611 and the stress granule marker eIF4G2-GFP were shifted to 37°C for 1 h. Although multiplePBs were formed in the arf1-11 mutant, we observed no induction of stress granules in the mutant or the control strain. (D) The numberof PBs in different temperature-sensitive mutants in components of the secretory pathway was determined after shift for 1 h to 37°C.All secretory mutants we analyzed displayed a multiple PB phenotype to a varying degree. (E) Quantification of the multiple PBphenotype in secretory mutants after a shift to the nonpermissive temperature. See B for details on the representation. Scale bars, (A,C, and D) 5 �m.

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Figure 2. Different stresses lead to induction of a different number of PBs. (A) Osmotic stress induces an increase in PB number in wild-typecells. Wild-type cells expressing Dcp2-GFP were grown in rich medium to early log phase at 23°C. Starvation was induced by incubating cellsin medium either lacking a carbon source (�D) or amino acids (�aa) for 15 min. To induce reductive or oxidative stress, 10 mM DTT or 0.4mM H2O2 were added to the cultures for 1 h. Spheroplasting by enzymatic digest (�Zymo) or treatment with calcofluor white (�CFW) wereused to induce cell wall stress. To induce osmotic stress, cells were harvested and either incubated in H2O or rich medium containing 0.5 MNaCl for 15 min. All stresses were applied at 23°C. Although cell wall stress did cause an increase in PB number, osmotic stress induced astrong multiple PB phenotype similar to arf1 mutants. (B) UPR is not generally activated in arf1 mutant alleles. Northern blot with a probeagainst HAC1 mRNA on total RNA extracted from ARF1 and arf1 cells grown at 23°C or shifted to 37°C for 1 h. The HAC1 cleavage productindicative of an active UPR is only observed in two of the mutant alleles. (C) The UPR components Gcn2 and Ire1 are not required forassembly of multiple PBs in arf1-11. Cells expressing Dcp2-GFP were deleted for either GCN2 or IRE1 and shifted to 37°C for 1 h. (D)Quantification of PBs in arf1-11 deleted for GCN2 or IRE1 after a shift to the nonpermissive temperature. See Figure 1B for details on therepresentation. (E) Quantification of the multiple PB phenotype in cells under osmotic stress and in the presence of ethanol. For the ethanolexperiment, wild-type or arf1-11 cells expressing Dcp2-GFP were shifted to 37°C for 1 h in the presence or absence of 1.6 M EtOH. No effectwas observed for wild-type, whereas the multiple PB phenotype in arf1-11 was rescued in the presence of EtOH. See Figure 1B for detailson the representation. (F) Wild-type cells expressing the stress granule markers Pub1-GFP or eIF4G2-GFP were grown in rich medium at 23°C

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increase in PB number similar to arf1 mutants (Figure 1E),indicating that this multiple PB phenotype is most likelyrelated to a general defect in secretion.

Hyperosmotic Stress Led to Numerous PBsHow do secretory transport mutants induce multiple PBs? Itis unlikely that this phenotype occurs in response to generalcellular stress. As has been reported before (Teixeira et al.,2005), PB formation is induced by starvation and underoxidation/redox conditions (Figure 2A). However, neitherstarvation nor ox/redox stress led to a similar increase in PBnumber in wild-type cells as observed for the arf1 or secre-tory pathway mutants; therefore, these mutants appear totrigger a different type of PB response. Secretory pathwaymutants may cause ER stress, which in turn would activatethe unfolded protein response (UPR). Alternatively, secre-tory transport mutants may prevent the correct targeting ofproteins to the plasma membrane and hence cause cell walland osmotic stress. First, we tested whether UPR is coupledto the formation of multiple PBs. The hallmark for the acti-vation of UPR is splicing of the transcriptional activatorHAC1 mRNA (Sidrauski and Walter, 1997). Shift of arf1-11to the restrictive temperature–induced UPR as indicated bythe cleavage of the HAC1 mRNA, which was also true forarf1-18, albeit to a lesser extent, but not for wild type andarf1-17 (Figure 2B), indicating that there is no direct corre-lation between UPR and PB formation. Moreover, deletionof IRE1, the endonuclease that splices HAC1 transcript at theER and senses unfolded proteins in the ER (Cox and Walter,1996; Kimata et al., 2006), or GCN2, the single eIF2alphakinase, which has been implicated in UPR (Patil et al., 2004),did not affect multiple PB formation in arf1-11 (Figure 2, Cand D). In contrast, when we challenged control or wild-type cells with cell wall stress, we detected an up-regulationof PB formation. More importantly, after application of os-motic stress by incubating the cells for a short period of time(15 min) in 0.5 M NaCl, a similar increase in the number ofPBs was observed as in the arf1 mutants (Figure 2, A and E).We also examined SG formation under the same conditionsto determine if these stresses were specific for assemblingmultiple PBs. Only 1–2 SG were induced under variousstresses, except for robust heat stress (46°C), using Pub1-GFP, eIF4G2-GFP, and eIF3B-GFP as markers (Figure 2F,Supplemental Figure 2B), indicating that this stress is PBspecific. The foci formed at 46°C represent SG because eIF3localizes to SGs after robust heat shock (Grousl et al., 2009).These data could indicate that in arf1 mutants the response

to osmotic or cell wall stress is activated, and that thisspecific stress response increases PB number. Our data areconsistent with the possibility that components of the cellwall or plasma membrane constituents do not reach theplasma membrane efficiently in arf1 and most secretorypathway mutants at the nonpermissive temperature, and thecells become more susceptible to osmotic stress. If this hy-pothesis was true, preventing signaling in the osmo-re-sponse pathway should rescue the multiple PB phenotype inarf1-11 mutants.

Neither the High-Osmolarity Glycerol Nor the Cell WallIntegrity Pathways Are Required for the Increase in PBNumber in arf1 MutantsThe major pathway activated in response to osmotic stress isthe high-osmolarity glycerol (HOG) mitogen-activated pro-tein (MAP) kinase signaling pathway (Van Wuytswinkel etal., 2000; Hohmann, 2002). The HOG pathway is at leastpartially repressed by addition of ethanol to the growth me-dium (Hayashi and Maeda, 2006). Strikingly, a very strongreduction in PB number was observed when arf1-11 cells wereshifted to the restrictive temperature in the presence of ethanol(Figure 2E), suggesting that the HOG pathway may be acti-vated in the arf1-11 mutant. Hog1 is not essential, and thereforewe could test the requirement for HOG signaling pathway inPB formation directly by deleting HOG1 in arf1-11. Surpris-ingly, the number of PBs was unchanged upon loss of Hog1pin arf1-11 at 37°C (Figure 2H, Table 2). Moreover, deletion ofthe osmo-sensor SHO1, or the HOG-dependent osmo-respon-sive transcription factor SKO1, did not interfere with PB for-mation in arf1-11 (Figure 2H, Table 2). The results indicate thatHog1 is not involved in PB assembly in secretory mutants. Thisis in agreement with recent data, suggesting that Hog1 mayplay a role in PB disassembly, and that PBs are formed in �hog1cells under mild and severe osmotic shock (Romero-Santacreuet al., 2009).

Another signaling pathway acting at the plasma mem-brane is the cell wall integrity pathway, which plays a role inthe progression through the cell cycle. The MAP kinaseSlt2p, also referred to as Mpk1p, is central to the cell wallintegrity pathway (Lee et al., 1993) and is phosphorylated inresponse to cell wall stress. We observed the phosphoryla-tion of Slt2 in arf1-11 cells after shift to the nonpermissivetemperature (Figure 2I). A low level of phosphorylation isalready observable in arf1-11 cells at 23°C and in wild-typecells at 37°C, but these levels may be too low to drivenumerous PB formation. Similar to Hog1, Slt2 is not essen-tial, and we could determine the number of PBs in an arf1-11�slt2 strain at 37°C (Table 2). As in the case of loss of Hog1,deletion of SLT2 did not reduce PB number in arf1-11 cells,showing that under these conditions neither the HOG northe cell wall integrity pathway is involved in PB formation.Moreover, the two pathways do not cooperate in PB induc-tion, because a deletion of both HOG1 and SLT2 did notreduce PB number in arf1-11 cells (Figure 2H, Table 2).Finally, we deleted the serine/threonine kinase Sli2p (alsoreferred to as Ypk1p), which is involved in the cell integrityand sphingolipid-mediated signaling pathway (Schmelzle etal., 2002). Again, deletion of SLI2 had no effect on PB numberin arf1-11 cells (Table 2). Similarly, treating arf1-11 cells withchlorpromazine, a membrane-permeable molecule thatbinds anionic lipids such as polyphosphoinositides andleads to translation attenuation (De Filippi et al., 2007) didnot change the amount of PBs (data not shown). Takentogether, these results argue that the increase in PB numberseen in arf1 or secretory mutants is independent of the majorplasma membrane signaling pathways.

Figure 2 (cont). to early log phase and then either incubated inH2O, rich medium containing 0.5 M NaCl, or medium lacking acarbon source (�D) for 15 min, or subjected to high heat shock (10min at 46°C). We did not observe a marked induction of stressgranules under conditions that induce multiple PBs; however,Pub1-GFP containing stress granules were induced by heat shock.(G) Quantification of SGs in the wild-type induced by variousstresses. See Figure 1B for details on the representation. (H) Plasmamembrane stress signaling pathways are not required for the as-sembly of multiple PBs. Key components of different signalingpathways were deleted in arf1-11 cells expressing Dcp2-GFP. Noneof the mutants resulted in a reduction of PBs in arf1-11 at 37°C. SeeTable 2 for quantification of all mutations tested. (I) Lysates weregenerated from wild-type or arf1-11 cells after a 1 h shift to 37°C andanalyzed by immunoblot to detect total Slt2 and Slt2 phosphory-lated in response to cell wall integrity signaling. The band markedwith the arrowhead corresponds to phospho-Slt2. A lysate of astrain deleted for SLT2 was loaded as a reference. Scale bars, (A, D,F, and G) 5 �m.

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Ca2� Induces Numerous PBsBecause hyperosmotic shock leads to a transient increase inintracellular Ca2� (Batiza et al., 1996; Matsumoto et al., 2002)and a Ca2�/calcineurin-regulated response is required forgrowth under high osmolarity and cell wall stress (Heath et al.,2004), we asked next whether Ca2� is required for the forma-tion of multiple PBs. Incubation of wild-type cells with Ca2�-induced rapid PB formation, to the same extent as hyperos-motic shock, within 10 min after CaCl2 addition (Figure 3, Aand B). Moreover, the effect was specific to Ca2� becauseadding MgCl2 had no effect on PB formation (Figure 3, A andB). If formation of multiple PBs depends on increased Ca2�

levels, arf1-11 cells should not accumulate PBs in the presenceof Ca2�-chelating agents. To test this hypothesis, we precul-tured the strains in the presence of low concentrations ofBAPTA before and during the temperature-shift. Under theseconditions, only 1–2 PBs were observed in arf1-11 (Figure 3, Cand D). Moreover, Ca2� was less potent in inducing PBs inARF1. Similar results were also observed in the gamma-COPmutant sec21-1 and the exocyst component mutant sec6-4 upongrowth in BAPTA and shift to 37°C (Figure 3, C and D),indicating that the increase in PB number in secretory mutantsis dependent on a change in intracellular Ca2�. Interestingly, incontrast to the PBs formed under glucose starvation or NaCl,Ca2�-induced PBs disappear rapidly over time, and after �30min only a few, if any, PBs could be detected (Figure 3, E andF). Most likely transporters are expressed at the plasma mem-brane, which extrude excess Ca2� to maintain ion homeostasis.This effect is not observed in secretory pathway mutants, inwhich PBs persisted, even after a prolonged shift to the non-permissive temperature. This observation is expected as deliv-ery of transporters to the plasma membrane is blocked in thesemutants (Figure 1D).

Elevated Ca2� Levels and Starvation Lead to TranslationAttenuation and PB Formation through Different PathwaysSecretory pathway mutants cause translation attenuation at thenonpermissive temperature (Deloche et al., 2004) and inductionof PBs is thought to correlate with translation attenuation (Sh-eth and Parker, 2003). The hallmark of translation attenuationis a loss of translating polysomes and a concomitant increase inthe monosome (80S) fraction in the cell. First, we established

that arf1-11 cells indeed attenuate translation by determiningtheir polysome profile after shift to 37°C (Figure 4, A and B).Next we asked whether the number of PBs in the cell could becorrelated with the strength of translational block becauseParker and Sheth (2007) proposed a model in which the rates oftranslation and degradation of mRNAs would be governed bya dynamic equilibrium between polysomes and mRNPs in PBs.Therefore, we recorded polysome profiles of wild-type cellsthat were either starved for glucose or incubated with Ca2� ora combination of both stresses (Figure 4, B and C). As shownpreviously (Ashe et al., 2000), glucose starvation causes a pro-nounced shift of ribosomes from the polysome fraction towardmonosomes. A similar change was observed in cells that weretreated with Ca2� or NaCl and in arf1-11, albeit to a lesserextent (Figure 4). According to the dynamic equilibriummodel, the weak translational repression induced by highCa2�, NaCl, or the arf1-11 mutant would yield multiple PBs,whereas the strong translational repression observed uponstarvation would result in 1–2 PBs. It is conceivable that themultiple PBs we observe would coalesce to 1–2 PBs that con-tain more RNA or represent more densely packed, maturedPBs, if translational repression was stronger. If this hypothesiswas correct, combining starvation and high Ca2� should leadto the formation of 1–2 PBs. However, combining high Ca2� orNaCl with starvation led to the almost complete absence ofpolysomes, and even the monosomes seemed to partially dis-assemble, but multiple PBs were observed under these condi-tions (Figure 4C). These results indicate that dynamic equilib-rium might not be sufficient to explain different PB numbersobserved under starvation and high Ca2� and suggests thepresence of at least two distinct pathways to generate PBs, oneof which functions in sensing low glucose levels, whereas theother responds to an increase in Ca2� levels.

Calmodulin Is Required for the Induction of PBs inSecretory Pathway MutantsThe next question was how the cell would sense the potentialintracellular Ca2� changes. A major player in Ca2� signaling iscalmodulin (Cmd1), which contains four EF hands, three ofwhich can coordinate Ca2�. The temperature-sensitive cmd1-3mutant does not bind Ca2� (Geiser et al., 1991). Therefore, totest whether calmodulin is involved in signaling to PBs, we

Table 2. PB phenotype of various mutants

Supernumerary PBs

in arf1–11 at 37°C in ARF1 � Ca2� PBs induced by starvation

No deletion � (9.15 4.16) � (8.89 3.5) � (2.16 1.01)�hog1 � (8.07 3.5) � (7.51 3.38) n.d.�sho1 � (7.66 3.62) � (5.52 2.37) n.d.�sko1 � (7.72 3.32) � (5.43 2.55) n.d.�slt2 � (5.96 4.47) � (7.64 3.28) n.d.�hog1 �slt2 � (6.8 5.18) � (7.48 2.72) n.d.�sli2 � (7.65 4.05) � (6.9 3.45) n.d.�cnb1 � (6.41 2.48) � (6.71 2.74) n.d.�cmk1 �cmk2 � (7.65 3.45) � (6.27 2.85) n.d.�cmk1 �cmk2 �cnb1 � (7.72 4.05) � (6.81 2.8) n.d.�hog1 �slt2 �cnb1 � (7.97 4.75) � (8.32 3.35) n.d.�pat1 � (1.93 1.79) � (2.02 2.42) � (1.2 0.99)�scd6 � (2.04 1.81) � (1.08 1.27) � (2.41 1.28)cmd1-3 n.d. � (2.02 1.84) � (2.16 1.07)

Values in parentheses are average number of PBs counted per cell 1 SD. A minimum 100 hundred cells from at least two independentexperiments were counted for each strain. n.d., not determined.

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replaced CMD1 by the cmd1-3 mutation in our Dcp2-GFP–expressing wild type and asked if cells would still accumulatePBs in the presence of Ca2�. The cmd1-3 mutant blocked for-mation of numerous PBs (Figure 5A), demonstrating that PBinduction upon elevated Ca2� levels requires functional cal-modulin; however, is calmodulin also required for PB induc-tion by other stresses? To address this question, we analyzedPB assembly after induction by glucose starvation or NaCladdition, but detected no difference between wild-type andcmd1-3 cells (Figure 5, A and B). Similarly, when we introduced

the cmd1-3 allele into the arf1-11 mutant, induction of multiplePBs was reduced at the nonpermissive temperature (Figure 5,C and D), indicating that the PBs formed in the secretorymutants do indeed correspond to PBs induced by Ca2�, butdiffer from those induced by hyperosmotic stress, althoughthey are indistinguishable by light microscopy.

To determine if one of the established Ca2� signalingpathways is required for PB induction, we screened throughmutants in the phosphatase calcineurin– and in calmodulin-dependent kinases (Table 2). Deletion of the catalytic sub-

Figure 3. Ca2� induces PB assembly to a similar extent as that of a secretion block. (A) Wild-type cells expressing Dcp2-GFP were treatedwith 200 mM CaCl2, MgCl2 or 12.5 mM EDTA for 15 min at 30°C, washed and inspected under the microscope. Only Ca2� treatment inducedmultiple PBs. (B) Quantification of PBs in wild-type cells treated with Ca2�, Mg2� or EDTA. See Figure 1B for details on the representation.(C) The Ca2�-chelator BAPTA prevents formation of multiple PBs in secretory transport mutants. Cells expressing Dcp2-GFP werepre-cultured in the presence or absence of 0.6 mM BAPTA and then shifted to 37°C for 1 h before inspection under the microscope. PBinduction was strongly reduced. (D) Quantification of PBs in secretory mutants treated with BAPTA after shift to the nonpermissivetemperature. See Figure 1B for details on the representation. (E) Time-course analysis of PB induction under various conditions. Wild-typecells expressing Dcp2-GFP were either harvested and resuspended in rich medium without a carbon source (�D), or treated with 200 mMCaCl2 or 0.5 M NaCl. After various time-points, samples were removed, fixed with formaldehyde, and analyzed under the microscope. PBresponse to Ca2�appears to be transient, whereas PBs induced by osmotic stress or starvation persist longer. (F) Quantification of PBs in thetime-course experiment. See Figure 1B for details on the representation. Scale bars, (A, C, and E) 5 �m.

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unit of calcineurin, CNB1, or of the calmodulin-dependentkinases, CMK1 and CMK2, in arf1-11 mutant or wild-typecells had no effect on PB induction by temperature-shift orCa2� treatment, respectively. Even concomitant loss ofboth calmodulin-dependent kinases and the regulatorysubunit of calcineurin (�cmk1 �cmk2 �cnb1) or of HOG1,SLT2, and CNB1 (�hog1 �slt2 �cnb1) did not inhibit for-mation of numerous PBs in arf1-11 cells at 37°C or in ARF1cells in the presence of Ca2� (Table 2). Taken togetherthese data indicate that the classical Ca2� signaling path-ways are not involved in inducing PB production, sug-gesting that calmodulin might act on PBs by an unknownmechanism.

Calmodulin is an abundant protein that performs a pleth-ora of functions in the cell and is localized to sites of polar-ized growth and the spindle pole body (Ohya and Botstein,1994; Spang et al., 1996). When we appended Cmd1 withGFP in arf1-11 and wild-type cells and induced PBs bytemperature-shift or Ca2�, respectively, we did not observeaccumulation of Cmd1-GFP into multiple foci, indicatingthat Cmd1 is not a bona fide component of PBs. It is impor-tant to note, that under these PB inducing conditions Cmd1-GFP was lost from the site of polarized growth, the smallbud tip, providing corroborating evidence that the Ca2�

levels might be elevated in secretory mutants (SupplementalFigure 3).

Pat1 and Scd6 Are Required for PB Assembly in SecretoryPathway MutantsThe above data indicate the existence of multiple parallelpathways by which PB formation can occur. The assembly ofPBs has been best characterized under starvation conditions(Parker and Sheth, 2007; Teixeira and Parker, 2007; Franksand Lykke-Andersen, 2008). Interestingly, no single knowncomponent of PBs was solely responsible for PB formation(Teixeira and Parker, 2007). Thus, if different pathways existthat lead to PB formation, one might be able to identify PBcomponents that are required for PB formation only underspecific conditions. To test this hypothesis, we tested two PBcomponents that are not strictly involved in PB formationunder starvation, namely Pat1 and Scd6 (Teixeira and Parker,2007). We chose Pat1 because it contains an EF-hand, whichmight coordinate a Ca2� ion. Pat1 binds, together with theLsm1-7 complex, to the 3� region of mRNAs, and is involved inPB formation. However, PBs are still formed in �pat1 cellsupon glucose starvation (Teixeira and Parker, 2007). Scd6 is anSm-like protein (Lsm) most likely involved in the regulation ofmRNA translation and/or degradation in PBs (Decker andParker, 2006) and was first described as a multicopy suppres-sor of a membrane transport defect (Nelson and Lemmon,1993). Deletion of PAT1 or SCD6 in wild-type cells stronglysuppressed PB assembly induced by Ca2� (Figure 6, A and B).

Figure 4. Translation attenuation and PB in-duction in response to different stresses. (A)Wild-type or arf1-11 cells expressing Dcp2-GFP were grown in rich medium to early logphase at 23°C and the shifted to 37°C for 1 h.Cells were either inspected under the micro-scope or subjected to polysome profile analy-sis. In arf1-11 cells, translation is attenuated.(B) Quantification of the PBs observed in Aand C. See Figure 1B for details on the repre-sentation. (C) Cells expressing Dcp2-GFP weregrown in rich medium to early log phase at23°C. Starvation was induced by resuspendingcells into medium lacking a carbon source(�D). Alternatively, cells were incubated inrich medium containing 0.5 M NaCl or 200mM CaCl2, or subjected to combinations ofthese stresses. After 15 min incubation at 23°Ccells were either inspected under the micro-scope or subjected to polysome profile analy-sis. Although starvation resulted in a markedincrease in the monosome/polysome ratio andinduction of few PBs, wild-type cells underosmotic stress or in the presence of Ca2� dis-played an intermediate increase in the mono-some/polysome ratio and had multiple PBs, aswas observed in the arf1-11 mutant shifted to37°C for 1 h. When starvation was combinedwith either osmotic stress or Ca2� treatment,polysomes were almost completely abolished,but cells had multiple PBs. Scale bars, (A andC) 5 �m.

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In agreement with previously published data, deletion ofPAT1 or SCD6 did not influence PB formation upon glucosestarvation of hyperosmotic shock, indicating that both pro-teins are required specifically for the formation of Ca2�-dependent PBs (Figure 6, A and B). Next, we investigatedthe effect of PAT1 or SCD6 deletion in arf1-11 and sec6-4 aftershift to the nonpermissive temperature. As expected, thesecells did not form multiple PBs (Figure 6, C and D); how-ever, most cells did contain a single PB, reinforcing the ideathat PB assembly is not strictly dependent on Pat1 or Scd6.When we appended Pat1 or Scd6 with GFP in wild-type andarf1-11 mutant cells and subjected them to Ca2� treatment orshift to 37°C, respectively, the GFP signal remained mostlycytoplasmic. However, multiple GFP foci formed underthese conditions, indicating that both proteins are compo-nents of the PBs we observe (Figure 6E).

Taken together, our data provide evidence that the mul-tiple PB phenotype in secretory pathway mutants is due tochanges in intracellular Ca2� and that Pat1 and Scd6 areinvolved in translating these changes into PB assembly.

Rescue of PB Formation in Secretory Pathway Mutants IsIndependent of Translation DerepressionGiven the relationship between PB formation and block oftranslation, we next wondered whether translation attenua-tion in the secretory mutant arf1-11 would also be rescued bydeletion of PAT1 or SCD6 or by introducing the cmd1-3

allele. We shifted cells for 1 h to the nonpermissive temper-ature and recorded the polysome profile. Interestingly, al-though deletion of PAT1 restored translation even abovewild-type levels, neither deletion of SCD6 nor the mutationin calmodulin influenced translation levels (Figure 6F), in-dicating that PB formation can be prevented independentlyof translation derepression.

PBs Are in the Vicinity of the ERPB formation in secretory pathway mutants requires Scd6.The Lsm Scd6 homologue in Caenorhabditis elegans, CAR-1, isrequired for ER organization (Squirrell et al., 2006). TheDrosophila homologue, trailer hitch, is localized close to ERexit sites, and is required for normal ER-exit site formation(Wilhelm et al., 2005). We therefore hypothesized that PBsinduced in secretory mutants localize to the ER. To test thishypothesis, we examined the colocalization of Dcp2-GFPfoci with an ER marker, Sec63-RFP, and found that theywere in close proximity to the ER, in both wild-type and arf1mutant cells shifted to 37°C (Figure 7A). Immunoelectronmicroscopy confirmed this observation (Figure 7C), andgold particles were found next to the ER in both mutant andwild-type cells after shift to 37°C or in wild-type cells understarvation. The labeling was specific, because no gold parti-cle accumulations were found in an untagged strain (Sup-plemental Figure 4). Neither Scd6 nor Pat1 were required forthe juxtaposition of PBs and ER (Figure 7B).

Figure 5. Calmodulin is required for the assembly of multiple PBs in the presence of Ca2� and in secretory mutants. (A) PBs were inducedin wild-type and cmd1-3 mutant cells expressing Dcp2-GFP by treatment with 200 mM CaCl2, 0.5 M NaCl, or incubation in rich mediumlacking a carbon source (�D) for 15 min at 23°C, or after a 1 h shift to 37°C. Although PB induction by osmotic stress or starvation was notaffected in the cmd1-3 mutant, a strong reduction of PB number was observed in the mutant after Ca2� treatment. (B) Quantification of PBsin the ARF1 cmd1-3 mutant induced either at 23°C or after a shift to 37°C for 1 h. See Figure 1B for details on the representation. (C) arf1-11cells expressing Dcp2-GFP that were deleted for CMD1 but carried a plasmid containing the cmd1-3 allele were shifted to 37°C for 1 h. PBnumber was reduced if compared with arf1-11 alone. (D) Quantification of PBs in the arf1-11 cmd1-3 mutant induced at 37°C. See Figure 1Bfor details on the representation. Scale bars, (A and C) 5 �m.

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We observed that the Dcp2-myc signal was organized inring-like structures, which had in all cases a diameter of �40to 100 nm (Figure 7D). Therefore, surprisingly, the PBs in-duced under various conditions share a strikingly similarorganization. To confirm this result, we determined the lo-calization of another PB component, the helicase Dhh1,which is not essential for PB formation (Teixeira and Parker,2007). Interestingly, Dhh1 accumulated in similar size foci,again close to the ER, suggesting that Dcp2 and Dhh1 arepart of the same spherical structure (Figure 7C), indepen-dent of the induction condition or the strain backgroundused. A spherical structure for PBs has been previouslyproposed (Kedersha et al., 2005; Teixeira et al., 2005; Wilc-zynska et al., 2005); however, the data underlying this hy-pothesis were obtained by light microscopy. Given the sizeof 40–100 nm of PBs, the resolution of a standard lightmicroscope does not allow a precise measurement. Interest-ingly, the size of the PBs was similar in wild type and arf1-11(Figure 7D). Therefore, PB number could potentially be cor-

related to the amount of mRNA that has to be silencedand/or to be degraded.

PBs Are Tightly Associated with the ERThe close proximity of PBs to the ER prompted us to testwhether PBs are bound to the ER. First, we performed adifferential centrifugation of total yeast cell lysate (Figure7E) and found the PB marker Dcp2-myc segregated into theP13 and the S13 fraction. We would not expect all Dcp2 to beefficiently depleted from the soluble pool, because we al-ways observed cytoplasmic Dcp2-GFP under the micro-scope. The accumulation of a PB component in the P13fraction could be explained either by its membrane associa-tion or simply by sedimentation of PBs at medium speedbecause of their large size and mass. To distinguish betweenthese possibilities, we performed a buoyant density centrif-ugation with the P13 fraction (Figure 7E). Membrane parti-cles would float, whereas large protein complexes shouldremain in the bottom of the tube. A fraction of Dcp2 floated

Figure 6. Pat1 and Scd6 are required for PB assembly in secretory transport mutants and upon Ca2� treatment. (A) PBs were induced inwild type, �pat1 or �scd6 mutant cells expressing Dcp2-GFP by treatment with 200 mM CaCl2 or 0.5 M NaCl, or after incubation in richmedium lacking a carbon source (�D) for 15 min at 23°C. Although PB induction by osmotic stress or starvation was not affected in thedeletion mutants, a strong reduction of PB number was observed after Ca2� treatment. (B) Quantification of PBs in the �pat1 or �scd6 strainscompared with wild-type. See Figure 1B for details on the representation. (C) Pat1 and Scd6 are required for assembly of multiple PBs insecretion mutants. Wild type, arf1-11, and sec6-4 expressing Dcp2-GFP and deleted for either PAT1 or SCD6 were shifted to 37°C for 1 h.Deletion of PAT1 or SCD6 abolished the multiple PB phenotype in secretion mutants. (D) Quantification of PBs in secretory mutants deletedfor PAT1 or SCD6 after a shift to the nonpermissive temperature. See Figure 1B for details on the representation. (E) PBs were induced inwild-type or arf1-11 cells expressing either Pat1-GFP or Scd6-GFP by incubation in rich medium lacking a carbon source (�D), treatment with200 mM CaCl2 for 15 min at 23°C, or a shift to 37°C for 1 h, respectively. Both Pat1-GFP and Scd6-GFP behaved similarly to Dcp2-GFP andwere found in multiple PBs after Ca2� treatment or in the secretory mutant after shift to the nonpermissive temperature. (F) arf1-11 cellsdeleted for PAT1 or SCD6, or carrying the cmd1-3 allele were shifted to 37°C for 1 h and their polysome profile recorded. Although all three strainsdo not induce multiple PBs under this condition, translation is derepressed only in the strain deleted for PAT1. Scale bars, (A, C, and E) 5 �m.

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to the same position in the gradient as the translocon com-ponent Sec61p, indicating that PBs do not only localize closeto the ER but are physically associated with the membrane(Figure 7E, 0/40 interphase). This localization of PBs to theER suggests the intriguing possibility that translation ofmRNA on one hand and mRNA silencing and degradationon the other hand occur in a spatially coordinated manner atthe ER.

DISCUSSION

We have characterized a pathway through which PBs areinduced in response to a block in secretion. This pathway isdifferent from PB induction under starvation. Moreover, we

found that not all stresses elicit assembly of PBs to the sameextent. Although we observed only 1–4 PBs per cell uponstarvation or exposure to oxidative stress, numerous PBswere formed in response to secretion defects or treatmentwith Ca2� (average 9–10). Although appearance of multiplePBs could also be induced by osmotic stress, this pathwaywas independent from Ca2�.

Thus far, no specific signaling pathway has been impli-cated in the formation of PBs. The common view is ratherthat a number of stresses cause attenuation of translationinitiation, and, as a consequence, mRNAs accumulate in thecytoplasm and passively aggregate into PBs (Andrei et al.,2005; Coller and Parker, 2005; Ferraiuolo et al., 2005). How-ever, when we combined stresses, the PB number did not

Figure 7. PBs associate with the ER. (A) Wild-type and arf1-11 mutant cells expressing Dcp2-GFP and the ER marker Sec63p-RFP wereshifted to 37°C for 1 h. In both control andarf1-11 cells, PBs were observed next to the ER.White arrowheads point toward a selection ofPBs close to the ER. (B) PBs were induced inwild-type cells expressing Dcp2-GFP and theER marker Sec63p-RFP and deleted for eitherPAT1 or SCD6 by incubating cells in rich me-dium without a carbon source for 15 min. In thecontrol as well as in the deletion strains, PBswere observed in proximity to the ER. Whitearrowheads point toward a selection of PBsclose to the ER. White bars (A and B), 5 �m. (C)Dcp2 and Dhh1 are part of spherical structuresin proximity to the ER. Wild-type and arf1-11cells expressing either Dcp2–9myc or Dhh1–9myc were shifted to 37°C for 1 h or incubatedin rich medium without a carbon source for 15min (�D) at 23°C. Cells were fixed and ana-lyzed by immuno-EM for myc. Clusters of goldparticles localized next to ER membranes. Scalebar, 200 nm. The inlet is a twofold magnifica-tion of the area surrounding PBs. White arrow-heads point to ER membranes; N marks a nu-cleus and SPB with a black arrowhead points toa spindle pole body in the nuclear envelope.(D) Determination of the approximate diameterof PBs. The largest distance between two goldparticles in a cluster was determined for Dcp2–9myc and Dhh1–9myc in wild-type and arf1-11cells. The size of the clusters did not changesignificantly between different conditions forthe two different markers. Each dot representsan individual PB, the horizontal line marks theaverage. (E) PBs are physically connected to theER. Yeast lysates of wild-type and arf1-11 cellsexpressing Dcp2–9myc were prepared after ashift to 37°C for 1 h and separated into a13,000 � g pellet (P13) and a correspondingsupernatant (S13). The pellet was subjected tobuoyant density centrifugation. A fraction ofDcp2–9myc cosedimented with the ER markerSec61p in the P13 fraction and floated withSec61p to the 0/40% sucrose interphase. Dcp2–9myc behaved similar in wild-type and arf1-11lysates.

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Molecular Biology of the Cell2636

correlate with the strength of the translational block, butrather with the type of stress encountered, indicating that PBnumber is regulated by an upstream signal dependent onthe stressor, but less dependent on the translational re-sponse. Interestingly, when other labs studied PB inductionunder starvation, loss of Dhh1 and Pat1 decreased the num-ber of PBs but corresponded with increased polysome-asso-ciated mRNA (Coller and Parker, 2005), implying that anequilibrium between translation initiation an PB formationcould exist. When secretory mutants were deleted for PAT1,we observed a similar phenomenon. However, when wedeleted SCD6 or introduced cmd1-3 into arf1-11, PB induc-tion was strongly reduced, but the attenuation of translationinitiation remained unchanged, indicating an uncoupling oftranslation initiation and PB formation. Therefore, mRNArelease from polysomes might not be the driving force for PBformation in secretory mutants.

The PBs induced in secretory transport mutants were verysimilar to PBs formed under starvation or heat stress asjudged by immunoelectron microscopy. Thus the increase innumber of PBs would suggest that more mRNA might haveto be silenced and eventually be degraded, if the stresspersisted over longer periods. In mammalian cells, PBs ap-pear to be highly dynamic structures in that they can quicklyexchange a subset of the constituents with the cytoplasm(Andrei et al., 2005; Kedersha et al., 2005). It is thereforeconceivable that the composition of PBs might vary depend-ing on the particular stress that induced their assembly.Individual PB proteins may also have different exchangerates and vary in the extent to which they can be exchanged.In fact, Dcp2 seems to be one of the less exchangeable PBconstituents (Aizer et al., 2008). Thus Dcp2 and Dhh1 levelsmay not change dramatically between different PB subtypes,which may therefore be indistinguishable by immunoelec-tron microscopy approach. Because the Ca2�-induced PBsare much shorter lived than the starvation-induced PBs, it isplausible that also a maturation pathway for PBs exists. Thehallmark of such a pathway would first be mRNA storageand later mRNA degradation.

The data we present here challenge the current prevailingmodel of PB assembly on various levels. First, differentstresses lead to a different number of PBs independent of thelevel of translation attenuation (Figures 2 and 4). Second, thediameters of Dcp2-myc and Dhh1-myc rings, which we as-sume represent PBs, do not significantly change under var-ious stresses (Figure 7). If PBs were mere mRNP aggregates,why would they be uniform in size? Third, the formation ofPBs after secretory transport block required functional cal-modulin, whereas Dcp2-GFP foci were still observed instarved cmd1-3 mutant cells subjected to starvation or os-motic stress (Figure 5). Finally, the Lsm-associated proteinPat1 and the Lsm homologue Scd6 were essential for PBformation in secretory pathway mutants or in Ca2�-treatedwild-type cells but not during starvation or NaCl treatment(Figure 6). Therefore our data indicate that parallel path-ways for PB induction must exist and that those pathwaysmay modulate the extent to which PBs are formed.

Here, we identified three novel modulators of the PBresponse: calmodulin, Scd6, and Pat1. The interaction part-ner for calmodulin in PB assembly remains elusive, butcalmodulin does not seem to act through CaM kinases as theconcomitant loss of CaM kinase 1 and 2 (�cmk1 �cmk2) hadno effects on PB formation.

Whether Cmd1, Scd6, and Pat1 act in parallel or whetherCmd1 is upstream of Scd6 and Pat1 remains unclear. TheEF-hand contained in Pat1 does not seem to be directlyinvolved in Ca2� sensing; in a strain in which we mutated

the putative Ca2� coordinating residues, multiple PBs werestill induced in response to Ca2� (unpublished results). Weenvision a pathway in which secretory pathway mutantswould trigger a change in intracellular Ca2�, and thischange would be sensed by calmodulin. Calmodulin mightthen either directly, or through Scd6 and Pat1, promote PBformation. The assembly of PBs induced by starvation orother stresses would follow a different, calmodulin-indepen-dent route.

ACKNOWLEDGMENTS

We are grateful to E. Schiebel, A. Diepold and members of the Spang lab fordiscussions, and to E. Hartmann and I. G. Macara for critical comments on themanuscript. S. Michaelis is acknowledged for providing the Sec63-RFP plas-mid, and we thank T. Davis (University of Washington, Seattle, WA), M. Hall(University of Basel, Basel, Switzerland), A. Nakano, R. Schekman, and E.Schiebel for reagents. This work was supported by the Boehringer IngelheimFonds (C.K.), the Werner Siemens Foundation (J.W.), the Swiss NationalScience Foundation, and the University of Basel.

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